Congenital gastrointestinal motility disorders arise as a consequence of anomalies in development of the enteric nervous system (ENS). While progress in defining the regulatory molecules and signaling pathways that govern ENS development has come from studies of spontaneous and engineered mouse mutants, our understanding of neuronal and glial lineage divergence in this system is still rudimentary. Human and mouse mutations demonstrate that Sox10 is critical for the neural crest (NC) progenitors that populate the ENS during development as haploinsufficiency or dominant negative forms of this transcription factor lead to aganglionic megacolon. However, little is known regarding the specific ENS cell types that derive from Sox10 precursors. Fundamental insight into the processes of ENS development and the complex cell interactions that can produce motility disorders may arise from detailed analysis of Sox10 NC derivatives. To evaluate the role of Sox10 within the context of normal ENS ontogeny, conventional gene disruption analyses that result in haploinsufficiency must be avoided. We propose to develop and apply transgenic approaches that harness Sox10 regulatory regions in conjunction with detailed immunohistochemical studies to determine the relationship of Sox10 to cell lineage divergence in the ENS. By comparative genomic sequence analysis we have identified potential cis-regulatory regions scattered throughout the Sox10 locus. Our initial analysis of 5' flanking sequence indicates that regulatory elements either within introns or at a distance from the gene must be required in vivo to recapitulate normal Sox10 expression. To generate tools for investigation of cell lineage pathways in the ENS, we will use BAC modification to localize the regulatory regions that confer expression of Sox10 in enteric NC. Modified Sox10 BACs driving expression of inducible CRE reporters will be deployed in transgenic mice as lineage tracing tools to investigate the relationship of Sox10 to divergence of neurons and glia in the ENS. Coupled with Sox10 immunohistochemistry (IHC) during embryonic migration and differentiation of enteric NC, these studies will construct a detailed picture of ENS lineage divergence. Our analysis will pioneer exploration of cell lineage pathways in the ENS and provide essential reagents for future functional analysis of Sox10 and other genes with potential relevance for ENS development. This proposal represents a distinct divergence from the PI's main research focus and qualifies for consideration under the "Innovative and Exploratory Research in Digestive Diseases and Nutrition" Program Announcement.